We identified 13 nuclear-localized long noncoding (lncRNAs), transcripts longer than 200 nucleotides that do not have functional protein coding capacity, that are essential for terminal erythroid differentiation from the mouse fetal liver erythropoietin-dependent CFU-E stage. All are highly upregulated during terminal red cell development and are expressed mainly or exclusively in erythroid cells; the promoters of most of these genes are targeted by the key erythroid transcription factors GATA1, TAL1, and/or KLF1. Three of these lncRNAs regulate adjacent genes: One enhancer transcript, alncRNA-EC7, is specifically needed for activation of the gene encoding BAND3 and another, elncRNA-EC3, is essential for upregulation of the KIF2A gene. shlncRNA-EC6, also called DLEU2, remains localized to its site of transcription in the nucleus, and the DLEU2 gene locus interacts with the putative promoters of at least 8 neighboring genes on the same chromosome. Expression of shlncRNA-EC6 is essential for normal downregulation of these genes during terminal erythroid proliferation and differentiation. Therefore, we hypothesize and will test that alncRNA-EC7 and elncRNA-EC3 establish an activating chromatin structure around their adjacent genes, whereas shlncRNA-EC6 establishes a repressive one. More generally this grant is focused on determining precisely how each of these 13 erythroid-specific lncRNAs regulates terminal red cell development. To this end, we will clone the full-length transcript of each lncRNA and determine whether each lncRNA transcript remains near its genetic locus. In parallel, we will identify all of the genomic DNA segments with which each lncRNA interacts, and determine whether, as we predict, interaction of the lncRNA genetic locus with other chromosome segments is dependent on expression of the lncRNA. We will identify the nuclear proteins bound to each erythroid lncRNA, and determine the functional association between erythroid-important lncRNAs and the proteins that specifically bind them during erythroid development. In parallel, we will determine the broad functions of these 13 lncRNAs in erythroid development. An overview of the requirement for each lncRNA will be done by assaying RNA knockdown cells for effects on terminal differentiation including cell proliferation, cell cycling, apoptosis, induction of erythroid important marker genes, and nuclear condensation. In parallel we will use deep sequencing to identify all mRNAs whose up- or down-downregulation during development is directly or indirectly dependent on induction of each lncRNA. Taken together, these experiments will enable us to formulate a specific hypothesis as to how each lncRNA regulates erythropoiesis and begin to test this hypothesis. Finally, we will confirm the roles of each of these 13 erythroid-important nuclear lncRNAs in vivo by generating knockout mice using the Cas9 system. We expect all to be embryonic lethal with severe anemia, and we will analyze in detail fetal and if need be adult erythroid development.